In the continuing effort to tap the vast, unexplored reaches of the earth's microbial and plant domains for bioenergy and environmental applications, the DOE Joint Genome Institute (DOE JGI) has announced its latest portfolio of DNA sequencing projects that it will undertake in the coming year.

The 44 projects, culled from nearly 150 proposals received through the Community Sequencing Program (CSP), represent over 60 billion nucleotides of data to be generated through this biodiversity sampling campaign—roughly the equivalent of 20 human genomes.

"The scientific and technological advances enabled by the information that we generate from these selections promise to take us faster and further down the path toward clean, renewable transportation fuels while affording us a more comprehensive understanding of the global carbon cycle," said Eddy Rubin, DOE JGI Director. "The range of projects spans important terrestrial contributors to biomass production in the Loblolly pine—the cornerstone of the U.S. forest products industry—to phytoplankton, barely visible to the naked eye, but no less important to the massive generation of fixed carbon in our marine ecosystems."

With new sequencing strategies coming on line at DOE JGI's Production Genomics Facility in Walnut Creek, Calif., Rubin said that the once daunting genome size of the Loblolly pine (Pinus taeda)—over 21 billion bases—is now becoming tractable. Loblolly pine is the most commonly planted tree species in America – accounting for about 75 percent of all seedlings planted each year.

"Its ability to efficiently convert CO2 into biomass and its widespread use as a plantation tree have also made Loblolly a cost-effective feedstock for cellulosic biofuel production and a promising tool in efforts to curb greenhouse gas levels through carbon sequestration," said Rubin. Because of the pine's enormous genome, the project will begin with a targeted effort to understand the structure of the pine genome. Led by Daniel Peterson of Mississippi State University, the project is intended to zero in on genes that can be used for molecular breeding programs to improve Loblolly as a biomass feedstock, carbon sequestration tool, and source of renewable, high-quality raw materials for lumber and pulp fiber.

The CSP selections range from these tall pines to not-so-sizable aquatic plants in duckweed—the smallest, fastest growing, and simplest of flowering plants. Greater Duckweed, Spirodela polyrhiza, is still relatively small at less than 10 millimeters. Nevertheless, its utility is manifold: as a biotech protein factory, toxicity testing organism, wastewater remediator, high-protein animal feed, carbon cycling player, as well as basic research and evolutionary model system.

"These plants produce biomass faster than any other flowering plant, and their carbohydrate content is readily converted to fermentable sugars by using commercially available enzymes developed for corn-based ethanol production," said Rubin. "Moreover, duckweed relates to all three of DOE JGI's mission areas: bioenergy, bioremediation, and global carbon cycling." Propagated on agricultural and municipal wastewater, Spirodela species efficiently extract excess nitrogen and phosphate pollutants. Duckweed growth on ponds effectively reduces algal growth (by shading), coliform bacteria counts, suspended solids, evaporation, biological oxygen demand, and mosquito larvae while maintaining pH, concentrating heavy metals, sequestering or degrading halogenated organic and phenolic compounds, and encouraging the growth of aquatic animals such as frogs and fowl. This project, submitted by Todd Michael of the Waksman Institute of Microbiology at Rutgers, The State University of New Jersey, unites the efforts of six institutions. The DOE JGI has selected several metagenomes to sequence—complex microbial communities that are isolated directly from the environment or reside inside of a larger organism. These leverage DOE JGI's pioneering expertise honed from previous studies of acid mine drainage and the termite hindgut—where samples yielded scores of different microbes, producing hundreds of enzymes with potentially useful industrial applications.

One such metagenome lurks inside of Bankia setacea, the giant Pacific shipworm. Shipworms, wood-boring marine bivalves, have been nicknamed "termites of the sea." These animals are capable of feeding solely on wood, utilizing a highly efficient system of symbiotic lignocellulose degradation that is biologically, functionally, and evolutionarily distinct from those found in termites, ruminants, and all other cellulose-consuming animals. Like termites, the ability of shipworms to consume wood depends on symbiotic bacteria that provide enzymes, including cellulases and other hydrolases critical for digestion of wood by the host and potentially valuable for commercial bioconversion of lignocellulose to ethanol. Analysis of the shipworm symbiont community metagenome will provide important insights into the composition and function of this unique lignocellulose degrading bacterial community and will allow valuable comparisons to the recently sequenced termite symbiont metagenome. Unlike termites, shipworms accomplish the complete degradation of lignocellulose with a simple intracellular consortium of just a few related types of microbes. The project was proposed by Daniel Distel of the Ocean Genome Legacy Foundation.

Another marine organism, Botryococcus braunii, is a colony-forming green microalga, less than 10 micrometers in size, that synthesizes long-chain liquid hydrocarbon compounds and sequesters them in the extracellular matrix of the colony to afford buoyancy. A type of B. braunii produces a family of compounds termed botryococcenes, which hold promise as an alternative energy source. Botryococcenes have already been converted to fuel suitable for internal combustion engines. Geochemical analysis has shown that botryococcenes, presumably from ancient B. braunii communities, also comprise a portion of the hydrocarbon masses in several modern-day petroleum and coal deposits.

While algae have been recognized for their role in carbon sequestration and for biofuels production, little information, either genetic or metabolic, has been reported for this particular organism. This project, led by Andrew Koppisch and colleagues from Los Alamos National Laboratory and five other institutions, will target the identification of specific metabolic pathways responsible for hydrocarbon synthesis to alleviate bottlenecks in biofuels production.

Other CSP 2009 projects include the following:

One metagenome project entails a sampling of the foregut of Opisthocomus hoazin—a leaf-eating Amazonian pheasant-like stinkbird, or hoatzin. A prehistoric relic, its unique fermentative organ harbors an impressive array of novel microbes, like that of cows and other ruminants. Instead of a rumen, stinkbirds possess a crop, an enlargement of the esophagus where the fermentation takes place—and the source of the stink. The characterization of its contents will likely lead to the identification of novel microbial enzymes that degrade plant cell walls.

Nanoflagellates, a group of marine microbes, prey on other microbes, such as bacteria and phytoplankton, for survival. These predatory protists play a critical role in marine carbon cycling. An International team of investigators led by Monterey Bay Aquarium Research Institute's Alexandra Worden will investigate the genetic mechanism behind the processes of predation, digestion, and biomass incorporation by protists that determine the fate of phytoplankton and bacteria to bridge the gap in our knowledge about this important player in the marine food web.

The most abundant source of carbon is plant biomass, composed primarily of cellulose, hemicellulose, and lignin. Many microorganisms are capable of utilizing cellulose and hemicellulose as carbon and energy sources, but a much smaller group of filamentous fungi has evolved with the ability to depolymerize lignin, the most recalcitrant component of plant cell walls. Collectively known as white rot fungi, they possess the unique ability to efficiently depolymerize lignin in order to gain access to cell wall carbohydrates for carbon and energy sources. Ceriporiopsis subvermispora rapidly depolymerizes lignin with relatively little cellulose degradation. The annotated gene set of C. subvermispora and comparative analyses with the lignin degraders P. chrysosporium and Pleurotus ostreatus (both sequenced by DOE JGI) will advance the understanding of these complex oxidative mechanisms involved in lignocellulose conversions. This project was proposed by Dan Cullen from the University of Wisconsin–Madison.

The CSP selections draw from all three branches of life: eukaryotes (such as plants and fungi), bacteria, and archaea. Desulfurococcus fermentans, isolated from the Uzon Caldera on the Kamchatka Peninsula, is the only known archaeon that breaks down cellulose and, unlike most known microorganisms that carry out fermentation, it produces hydrogen in the presence of hydrogen while fermenting cellulose and starch without experiencing an inhibition of growth. A comparative genomics investigation of Desulfurococcus species will resolve the finer details that distinguish proton reduction (producing hydrogen) from sulfur reduction in fermentative archaea and help to define the evolutionary and metabolic relationships of the Desulfurococcus species with their archaeal relatives. The project's principal investigator is Biswarup Mukhopadhyay of the Virginia Polytechnic Institute.

Among the holy grails of biofuel production is the perfect concoction of enzymes capable of rendering complex biomass into fuel by a process known as simultaneous saccharification and fermentation (SSF). Hansenula polymorpha strain NCYC 495 leu1.1 is a yeast capable of fermenting xylose (five-carbon sugar), glucose (six-carbon sugar), and cellobiose (a unit of two condensed glucose molecules) to ethanol at high temperatures (45° C), thus holding promise for the SSF process. Commercially feasible SSF technology has not yet been developed because of the absence of a robust organism capable of fermentation at high temperatures. Sequencing of H. polymorpha will enable the identification of the limiting steps in the fermentation pathway from xylose to ethanol. The project was proposed by Andriy A. Sibirny of the Ukraine's National Academy of Sciences and Rzeszów University in Poland.

Another key barrier to economical cellulosic biofuel production is the cost of enzymes for the degradation of cellulosic biomass. Currently, the cellulases used in pilot cellulosic ethanol plants are produced by fungi, in many cases Trichoderma reesei strain Qm6a (whose genome sequence analysis was published in Nature Biotechnology by DOE JGI and collaborators). The widespread use of T. reesei in cellulase production underscores the importance of this organism and the need for understanding the mechanisms behind enzyme secretion. This proposal, by Scott Baker from DOE JGI partner Pacific Northwest National Laboratory with contributors from Technical University of Vienna and biofuels industry players Verenium and Novozymes, will bridge the current gap in industrial fungal enzyme production research by sequencing five T. reesei strains with varying levels of cellulase production and derived from strain Qm6a with the purpose of characterizing the cellular machinery behind enzyme secretion.

The use of microbes to directly generate electricity from the biodegradation of waste organic matter in microbial fuel cells is a technology that shows great promise. Caroline S. Harwood of the University of Washington has proposed the sequencing of the electricity-generating photosynthetic bacterium Rhodopseudomonas palustris strain DX-1 to help highlight the mechanistic basis for this unusual biological property. This project will add to the growing literature describing the complexity of this genus by complementing the six other strains of R. palustris that have been sequenced to date by DOE JGI.

A census of subsurface microbial communities at the Hanford Site adjacent the Columbia River has been proposed by Allan Konopka and a multidisciplinary research group at Pacific Northwest National Laboratory. As part of initial site characterization efforts, a deep borehole will be drilled and core samples will be subjected to detailed microbiologic and geochemical analyses to address microbial ecology hypotheses and determine the composition and activity of subsurface microbial communities in microenvironments and across transition zones. Microenvironments are small domains within larger ones that exert a disproportionate influence on subsurface contaminant migration.

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